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The Laboratories for Reproductive Biology and the Departments of Pediatrics and Cell Biology and Anatomy (P.P.), University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7500
Address all correspondence and requests for reprints to: Frank S. French, Laboratories for Reproductive Biology, CB 7500, 382 Medical Sciences Research Building, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7500. E-mail: fsfrench{at}med.unc.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(24) interact with their
steroid activated ligand-binding domains. However, in AR, AF2 is
relatively weak compared with AF1 (13, 18, 19). In
cotransfection assays, the AR DNA and ligand binding domain fragment
lacks transcriptional activity in the absence or presence of androgen
(13, 19), whereas the N-terminal and DNA binding domain
exhibits strong constitutive activity (25). Nuclear receptors, like many other transcription factors, bind DNA as homo- or heterodimers (6, 26). AR homodimerization is enhanced markedly in the presence of androgen response element (ARE) DNA and is required for formation of a stable AR-ARE complex (27, 28). Dimerization of AR occurs through a DNA binding domain interface and antiparallel interactions between the N- and C-terminal domains (20, 28). Nuclear receptors increase the transcription rate of RNA through interactions with coactivators and the general transcription machinery (29, 30, 31, 32, 33). Transcriptional repression of specific genes is relieved through receptor binding of histone acetyltransferase coactivators and other chromatin remodeling factors (34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44) that increase the accessibility of nucleosomal DNA to the transcription complex. Herein we identify and characterize the functional effects of a human AR coactivator identical to the human thyroid receptor activator molecule TRAM-1 (45), a member of the SRC-3 subgroup of human steroid receptor coactivator-1 (SRC-1) family of p160 coactivators (31, 34, 46, 47, 48, 49), which includes human AIB1 (50), ACTR (51), and RAC3 (52). TRAM-1 and SRC-1a have 50% sequence identity and are expressed in a wide variety of tissues, although at different levels.
AR is expressed in specific cell types in testis, namely the cells that produce androgens and mediate androgen regulation of spermatogenesis: Leydig cells, peritubular cells, and Sertoli cells, respectively (53, 54, 55). Here we compare TRAM-1/SRC-3 and SRC-1a with respect to enhancement of AR transactivation and demonstrate SRC-3 expression in human seminiferous tubular cells that mediate androgen receptor regulation of spermatogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Full-length SRC-1a was excised from the vector pCR3.1 hSRC-1a (provided by Dr. Ming-Jer Tsai, Baylor College of Medicine, Houston, TX) using Pme1. The excised DNA was blunt ended with Klenow enzyme (Life Technologies, Inc., Rockville, MD) and ligated into the EcoR5 site of pSG5.
All constructs were confirmed by automated sequencing using a Perkin-Elmer Corp. Model 377 DNA sequencer.
Yeast two-hybrid screening
The vector pBDGAL4CAM expressing the GAL4 DNA binding domain
fused with human AR codons 481–919, which include the
carboxyl-terminal region of the N-terminal domain, DNA and
ligand-binding domains, was used as bait in yeast two-hybrid screening
of a HeLa cell complementary DNA (cDNA) library cloned into pGADGH
(CLONTECH Laboratories, Inc., Palo Alto, CA) (gift
of Dr. Yue Xiong, University of North Carolina-Chapel Hill). Yeast
strain Hf7c cotransformed with HeLa cell cDNAs was plated on
synthetic medium without Leu, Trp, His, and with addition of 1
µM DHT and 5 mM 3-amino-1,2,4 triazole (3AT).
Yeast colonies were picked for ß-galactosidase (blue-white) assay
after 5 to 7 days. cDNAs from yeast colonies that showed a blue color
consistently were rescued using standard procedures. Rescued HeLa cell
cDNA or control (lamin) expression vectors together with the AR bait
vector were used to transform yeast for testing of DHT dependency and
steroid specificity of AR interactions with recombinant HeLa cell
proteins. cDNA clones showing an androgen dependent AR interaction
stronger than the control were analyzed further in the liquid
ß-galactosidase assay.
Yeast liquid ß-galactosidase assay
Y190 yeast transformed with pBDGAL-AR (aa 481–919) and
pGADGH-TRAM-1 or pBDGAL-AR (aa 481–919) alone were incubated with
different steroids overnight at 30 C in 2 ml selective medium lacking
Trp and Leu, or lacking Trp alone with yeast containing only AR. After
incubation for 24 h at 30 C, YPD medium (8 ml) containing the same
concentration of steroid was added to each assay tube and incubation
was continued at 30 C for 3 h. Liquid ß-galactosidase assays
were performed according to the CLONTECH Laboratories, Inc. protocol.
Glutathione S-transferase-AR binding of [35S]
TRAM-1
GST-AR aa 1–660 and 544–634 were expressed in
Escherichia coli. Bacteria transformed with pGST-AR
fragments and cultured overnight at 37 C were diluted 1:10 in fresh LB
medium and incubated with shaking. After 2 h, IPTG was added to
final concentration of 1 mM and incubated with
shaking at 30 C for 3 h. Bacteria were collected by
centrifugation, and the fusion protein extracted 3 times by sonication
in buffer A (PBS containing 1 mg/ml BSA and 0.5% NP40, pH 7.4). GST-AR
(aa 624–919) was expressed from baculovirus in SF9 cells (vector
provided by Elizabeth M. Wilson, University of North Carolina, Chapel
Hill, NC). Full-length TRAM-1 cDNA was cloned into pSG5 and
[35S] TRAM-1 synthesized in the presence or
absence of 0.1 µM DHT using the TnT Quick
Coupled Transcription/Translation system kit (Promega Corp., Madison, WI). GST-fusion proteins were incubated 1 h
at 4 C with 20 µl glutathione-Sepharose beads (Amersham Pharmacia Biotech, Piscataway, NJ) in buffer A with or
without 0.1 µM DHT. After the incubation, beads
were washed three times with 1 ml buffer A at 4 C.
[35S] TRAM-1 was added and incubated 1 h
at 4 C. Washing with buffer A was repeated three times as above. SDS
buffer, 50 µl, was added and boiled 5 min. Supernatant proteins were
separated by SDS-PAGE in 8% gels. Gels were dried and autoradiography
performed with Kodak (Rochester, NY) X-OMAT film.
Immunohistochemistry
Human testicular tissue was obtained from an 81-yr-old man who
had received no previous therapy for prostate cancer before
orchiectomy. The tissue was fixed in Bouin’s fluid and embedded in
paraffin using standard procedures. Eight-micrometer sections were cut
and mounted on glass slides. Before immunostaining, endogenous
peroxidase was blocked (methanol + 5%
H2O2, 30 min at room
temperature) and antigen retrieval was carried out by microwave
treatment in 0.01 M (pH 6.0) citrate buffer. The slides
were immunostained according to the double PAP procedure as described
by Ordronneau et al. (56). Goat antirabbit IgG
serum absorbed against human proteins was obtained from Antibodies Inc.
(Davis, CA), donkey antigoat IgG and rabbit peroxidase-antiperoxidase
complex from Jackson ImmunoResearch Laboratories, Inc.
(West Grove, PA) and diaminobenzidine from Aldrich, Inc.
(Milwaukee, WI). As controls for specific staining, rabbit antiserum
was preadsorbed with the peptide antigen (100 µg/ml) and preimmune
serum from the same rabbit was substituted for immune serum.
Antibody to TRAM-1 was raised in a rabbit against carboxyl-terminal aa 1404–1424 and affinity purified using the peptide antigen. Affinity purified antibodies and unpurified antiserum showed little difference in immunostaining. Antibodies were tested by Western immunoblots of recombinant TRAM-1 expressed in COS cells (57) and immunoblotting of human testis protein extracts (58). Optimal antiserum dilution for immunostaining was 1:1500.
Western blot
Testis from a 75-yr-old man was stored in liquid nitrogen. One
gram of testis was pulverized in liquid nitrogen, resuspended in 3 ml
ice cold RIPA buffer (pH 7.4) containing protease inhibitors (0.5
mM phenylmethylsulfonyl fluoride, 10 µM
pepstatin, 4 µM aprotinin, 80 µM leupeptin,
and 5 mM benzamidine). The tissue was homogenized 15 sec
using a Polytron and sonicated 4 times, 5 sec each. After 30 min on
ice, the homogenate was centrifuged 15 min at 4 C and supernatant
collected. Centrifugation was repeated, and 250 µl aliquots of
supernatant protein were precleared by incubation 1 h with 25 µl
Pansorbin followed by centrifugation. Each cleared supernatant was
incubated at 4 C overnight with 25 µg affinity purified TRAM-1
antibody. Pansorbin, 25 µl, was added and the incubation continued
for 2 h at 4 C. Proteins were separated by 6% SDS-PAGE,
electroblotted to a nitrocellulose membrane and SRC-3 detected by
enhanced chemiluminescence (58). Peptide competition of
TRAM-1 antibodies was as described above.
Transient cotransfection assay
Cotransfection assays using monkey kidney CV1 cells were
performed as described previously (59, 60). In brief, 2.5
µg mouse mammary tumor virus long terminal repeat-luciferase reporter
vector (MMTV-Luc) provided by S. M. Hollenberg and R. M.
Evans (The Salk Institute, La Jolla, CA) and 0.1 µg human androgen
receptor (pSGhAR) expression vector were transfected without or with
coactivator cDNA, also in pSG5. Transfections with the recombinant pSG5
coactivator expression vector were compared with transfections of an
equal weight of pSG5 vector expressing the corresponding antisense RNA.
In other control plates, cells were transfected with an equal weight or
equimolar amount of empty pSG5 vector. DNA was transfected into
approximately 75–80% confluent CV1 cells in 6 cm culture dishes using
the CaPO4 method. After 4 h at 37 C, cells
were exposed to 15% glycerol for 4 min. and incubated in DMEM-H
without phenol red and serum in the presence or absence of steroid.
Medium was removed and fresh medium added after 20 h and the
incubations continued for a total of 40 h. The cells were
harvested using lysis buffer (Ligand Pharmaceuticals, Inc., San Diego, CA) and luciferase activity measured in
a luminometer. Data points were obtained in triplicate. Results shown
are representative of at least four different assays and include more
than one preparation of each plasmid.
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Results |
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The AR binding protein is one of a subgroup of human p160
coactivators
A database search using the GCG sequence comparison program
revealed sequence similarity with a subgroup of nuclear receptor p160
coactivators with a basic helix-loop-helix (bHLH) domain in the
N-terminal region, a centrally located nuclear receptor interaction
domain (RID) and a glutamine rich sequence in the C-terminal region
(Fig. 1
). The 3.3-kb cDNA sequence was
found to be identical to the cDNA sequence of thyroid receptor
activator molecule, TRAM-1, a 160-kDa, 1424-aa thyroid hormone receptor
(TRß) coactivator cloned from human pituitary and 293 cells
(45). TRAM-1 is the only member of the group that contains
an insertion of four amino acids (Gln-Val-Ser-Ser) residues 1213–1216.
It is otherwise identical to the cDNA sequence of human amplified in
breast cancer-1 (AIB1) identified as a coactivator for ER
(50) (Fig. 1). TRAM-1 and AIB1 are similar to receptor
associated coactivator-3 (RAC3) cloned from human brain
(52) but RAC3 has 26 instead of 29 Qs in the C-terminal
domain and in the bHLH/PAS domain contains the amino acids EA in place
of DG at residues 321, 322. TRAM-1 is also similar to ACTR
(51), which was cloned from human leukemia cells. However,
ACTR contains a 10-amino acid insertion in the bHLH/PAS domain and
deletion of 15 amino acids C-terminal to the RID (Fig. 1). ACTR also
contains R instead of G at residue 460, HG instead of QA at residues
1183, 1184, and 26 instead of 29 consecutive Qs. This subgroup of human
p160 coactivators was referred to as hSRC-3 (49). The
mouse SRC-3 gene was identified recently and shown to span more than 38
kb and contain 19 exons. SRC-3 isoforms are believed to represent
splice variants of the same gene (61).
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). We analyzed
further the steroid specificity of AR activation in yeast with
estradiol (E2), progesterone (P),
dehydroepiandrosterone (DHEA), hydroxyflutamide (OH-FL)
and dexamethasone (Dex) (Fig. 2B). At concentrations of 1
µM, none of these steroids induced a detectable AR (aa
481–919) interaction with TRAM-1. Similar results were obtained using
yeast strain Hf7c. These results indicate that the interaction between
the AR and TRAM-1 in yeast is androgen dependent.
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), full-length
[35S] TRAM-1 bound an AR fragment containing
the carboxyl-terminal region including hinge and ligand binding domain
(aa 624–919) (Fig. 3A) and an N-terminal and DNA binding domain
fragment (aa 1–660) (Fig. 3B), but binding to the DNA binding domain
(aa 544–634) was not detected. A major portion of the AR hinge region,
which includes aa 628–670, was present in the N-terminal and DNA
binding domain fragment (aa 1–660) as well as the ligand binding
domain (aa 624–919) but not in the DNA binding domain (aa 544–634).
These results suggest that TRAM-1 binds both the N-terminal and ligand
binding domains of AR; however, they do not exclude that the hinge or
DNA binding domain influences this binding.
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) and immunostained protein is
referred to as SRC-3. SRC-3 was present in nuclei of Sertoli cells and
peritubular myoid cells (Fig. 4A)
cell-types that also express AR (57). Thus AR and SRC-3
are coexpressed in the cells that mediate androgen regulation of
spermatogenesis. In contrast to AR, which in the germinal epithelium is
confined to Sertoli cell nuclei, SRC-3 is in spermatogenic cells in all
stages of development, where it might also regulate the activity of
other nuclear receptors. Similar results were obtained in rat and mouse
testes (not shown).
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). Similar immunostaining
results were obtained using peptide affinity purified antibody.
Staining was eliminated by serial dilution of antiserum, and no
staining was observed with preimmune serum. On a Western blot, the
antiserum (1:1500) reacted strongly with recombinant
TRAM-1 expressed in COS cells, and staining of the TRAM-1 band was
negative with the same peptide preadsorbed antiserum that was used as
control for immunostaining testis. Moreover, Western blots of human
testis protein extracts (Fig. 5) using
affinity purified antibody demonstrated a protein band that
corresponded to recombinant TRAM-1 expressed in COS-1 cells and was
eliminated by competition of antibody with the peptide antigen. Other
bands on the Western blot were not eliminated by peptide
competition.
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TRAM-1 is a coactivator of ligand-dependent AR and GR
transactivation
In initial tests of transcriptional coactivator function, we used
the partial TRAM-1 (pTRAM-1) cDNA isolated by two-hybrid screening.
pSG5-pTRAM-1 (codons 459-1424, see Fig. 1) was cotransfected into CV1
cells together with full-length AR (pSG5-AR, 0.1 µg), and a mouse
mammary tumor virus long terminal repeat-luciferase reporter vector
(MMTV-Luc, 2.5 µg). DHT, 0.1 nM, stimulated a 145-fold
increase over the no DHT background luciferase activity in cells
cotransfected with 1 µg pSG5-pTRAM-1 as compared with 26-fold with 1
µg pSG5 empty vector and 51-fold with AR and MMTV-Luc alone (Fig 6). The fold increase in the presence of
TRAM-1 was 5 times greater than with an equal weight of pSG5 parent
vector and 3 times greater than with AR and MMTV-Luc alone.
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). The fold increase in luciferase activity in
the presence of pTRAM-1 was 8 times higher than the fold increase with
an equal amount of pSG5 empty vector and 4 times higher than with GR
and MMTV-Luc alone.
Because TRAM-1 mRNA is expressed endogenously in CV1 cells
(63), we used a full-length TRAM-1 antisense expression
vector to balance transfections of full-length TRAM-1 sense expression
vector with a control DNA of equal weight and molarity. In addition,
translation of endogenous SRC-3 mRNA may have been inhibited by
antisense TRAM-1; however, this was not determined. DHT (0.1
nM) stimulated a 675-fold increase in luciferase activity
over the no DHT background in cotransfections with 5 µg pSG5-TRAM-1
sense DNA as compared with a 14-fold increase with 5 µg pSG5-TRAM-1
antisense cDNA and 59-fold with pSG5-AR and MMTV-Luc alone (Fig. 7). The fold increase in luciferase
activity in the presence of TRAM-1 was 48 times higher than the fold
increase attained with antisense TRAM-1 and 11 times higher than the
activity with pSG5-AR and reporter gene alone (Fig. 7).
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). DHT (0.1
nM) stimulated a 378-fold increase in luciferase activity
over the no DHT background with 5 µg pSG5-SRC-1 sense cDNA compared
with 114-fold with 5 µg pSG5-SRC-1 antisense cDNA and 59-fold with
pSG5-AR and MMTV-Luc alone. The fold increase in luciferase activity in
the presence of SRC-1 was 3 times higher than with antisense SRC-1 and
6 times higher than with AR and reporter gene alone.
Full-length TRAM-1 and SRC-1 also enhanced the transcriptional activity
of GR (Fig. 7). Dexamethasone (10 nM) stimulated a 240-fold
increase in luciferase activity over the no hormone background with 5
µg pSG5-TRAM-1 sense as compared with 33-fold with 5 µg pSG5-TRAM-1
antisense and 104-fold with GR and MMTV-Luc alone. The fold increase in
luciferase activity with TRAM-1 was 7 times higher than with antisense
TRAM-1 and 2 times higher than with GR and reporter gene alone.
In the presence of 5 µg pSG5-SRC-1 sense, 10 nM
dexamethasone stimulated a 211-fold increase over the no hormone
background compared with 153-fold with 5 µg pSG5-SRC-1 antisense and
104-fold with GR and MMTV-Luc alone. The fold increase in luciferase
activity was 2 times higher than with GR and reporter gene alone and
1.4 times higher than with SRC-1 antisense. In similar assays, addition
of control empty pSG5 vector DNA (5 µg) instead of the antisense
vector resulted in a 70% reduction of the activity of AR or GR and
reporter gene alone (data not shown). Thus when transfections with GR
in Fig. 7 were balanced with pSG5 empty vector instead of antisense
vector, both TRAM-1 and SRC-1 caused 7- to 8-fold increases in
transcription relative to the activity of GR and reporter gene with 5
µg pSG5 empty vector.
In CV1 cells the level of TRAM-1 mRNA determined by Northern hybridization appeared several times higher than that of SRC-1 mRNA (63), suggesting that higher endogenous TRAM-1 mRNA in CV1 cells may have accounted for the greater inhibitory effect of antisense TRAM-1 than antisense SRC-1 on AR and GR transactivation. In contrast to antisense TRAM-1 that decreased DHT-dependent AR induction of reporter gene transcription, antisense SRC-1 caused a 1.5- to 2-fold greater increase in luciferase activity when added to AR and reporter gene alone. We have observed similar effects with other cDNAs cloned in reverse orientation into pSG5 expression vectors but do not know if this is a DNA effect or an effect of the expressed antisense RNA.
TRAM-1 activates the AR N-terminal domain AF1
TRAM-1 and other p160 coactivators including SRC-1 contain a
receptor interaction domain with LXXLL motifs (Fig. 1) shown by
pull-down and two-hybrid assays to interact with AF2 in nuclear
receptor ligand binding domains (46, 64, 65, 66). SRC-1
interacts also with the AF1 region of the progesterone receptor
N-terminal domain (46). Because the AR N-terminal and DNA
binding domain (aa 1–660) has constitutive transcriptional activity
(13, 25), we compared the effects of TRAM-1 and SRC-1 on
the activity of this AF1 containing AR fragment (Fig 8). In cotransfection assays with
pSG5AR1–660 (10 ng) and MMTV-Luc (2.5 µg), pSG5-TRAM-1 stimulated a
9.4-fold increase and pSG5-SRC-1 a 3.6-fold increase above the level of
luciferase activity in assays with AR 1–660 and reporter gene alone.
In control transfections in which the sense vector was replaced by an
equal weight of antisense pSG5-TRAM-1 or SRC-1, luciferase activity was
50% less than with AR 1–660 and MMTV-Luc alone (not shown). Thus,
fold increases with TRAM-1 and SRC-1 were 19 and 7 respectively when
compared with increases in the presence of antisense vectors.
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Steroid specificity of TRAM-1 enhanced AR transactivation in
mammalian cells
Among the naturally occurring steroids in mammals, AR has highest
affinity for DHT (Kd 0.1 nM) but
binds estradiol and progesterone with lower affinity (67).
We tested whether the AR transcriptional enhancing effect of TRAM-1 is
specific for DHT or occurs also with other sex steroids capable of
binding and activating AR (Fig. 9). In
the CV1 cell cotransfection assay using pSG5-AR and MMTV-Luc with 3
µg pSG5-TRAM-1 sense vector, 0.1 nM DHT stimulated a
399-fold increase over the no DHT background luciferase activity
compared with 20-fold with 3 µg pSG5-TRAM-1 antisense vector and
20-fold with AR and reporter gene alone. The fold increase in
luciferase activity with sense TRAM-1 vector was 20 times higher than
the fold increase in the presence of antisense TRAM-1 or AR and
reporter gene alone (Fig. 9). Little or no effect was observed in the
presence of 0.1 nM progesterone, estradiol, or
hydroxyflutamide. These results indicate that TRAM-1 coactivation is
specific for DHT activation of AR within the steroid concentration
ranges that occur in peripheral tissues.
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Estradiol is produced in testis but is not likely to influence AR activation. The concentration of estradiol in testicular venous blood is 4–8 nM (73, 74), whereas testosterone is approximately 2400 nM (74).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p160 coactivators have a number of structural features in common. The
N-terminal region contains a potential basic helix-loop-helix
(51), which has an activation function in SRC-1
(46) and two Per-AhR-Sim domains that are found in several
nuclear proteins, including Peroid, aryl hydrocarbon receptor and
single minded (75). However, this region does not appear
to be required for function as a coactivator with AR or GR. A receptor
interaction domain contains three LXXLL motifs (51, 62, 64, 76), each of which predicts an amphipathic 
-helical structure
with the conserved leucines forming a hydrophobic surface. Both
N-terminal (aa 1–780) containing the receptor interaction domain and
C-terminal (aa 877-1424) regions of TRAM-1 interacted with TR
(45). A single LXXLL motif is present in the C-terminal
region of TRAM-1; however, a C-terminal fragment (aa 800-1215)
containing this motif did not bind TR or RXR indicating residues in
addition to the LXXLL motif were required for the C-terminal binding
(45). C-terminal SRC-1 bound the PR ligand binding domain
and was one of several regions of SRC-1 that interacted with PR
(46).
The AF2 domain of RXR was shown to be a site of p160 coactivator interaction. ACTR binding to RXR in GST-fusion protein pull-down assays was either abolished or diminished by AF2 mutations (51). Similarly, SRC-1 binding to TR was abolished by an AF2 mutation; however, the same mutation had less effect on the binding of TRAM-1 suggesting it may have sites of interaction outside the RXR AF2 domain (45).
RAC3 was shown to have an intrinsic activation function in a mammalian cell one-hybrid assay when fused to the GAL-4 DNA-binding domain (52). In similar assays with ACTR, the activation function was C-terminal to the receptor interaction domain (within aa 827-1412) and coincided with the region that bound CBP and pCAF indicating that these coactivators account for the transactivation function of this region. The C-terminal region of ACTR had intrinsic histone acetyltransferase activity. Similarly, the TRAM-1 C-terminal region (aa 800-1215) but not N-terminal region containing the receptor interaction domain bound CBP and pCAF (45, 51), suggesting that TRAM-1 and CBP form a transcription complex with TR. Histone acetyltransferase domains are essential to the coactivator functions of CBP and pCAF (77). The role of C-terminal repeated glutamines in the hSRC-3 subgroup of p160 coactivators remains to be determined. However, glutamine repeats occur commonly in transcription factors and have been shown to modulate transcriptional activity in cell-free transfection assays (78). Glutamine repeats present in the N-terminal region of the human AR (1, 2, 3, 4) are polymorphic and range between 11–31 with a mean of 22 ± 2 in the normal population (79).
Our data showing that TRAM-1 and SRC-1 enhance the constitutive transcriptional activity of the AR N-terminal and DNA binding domain fragment is consistent with the concept that AF1 is the major activation domain of AR. Langley et al. (19, 20) discovered that the N-terminal region of AR interacts strongly with its ligand activated C-terminal region. This interaction was abolished by deletion of N-terminal aa 14–150 or 339–499 but was less affected by deletion of residues 142–337 that make up a major transactivation unit (AF1) within the N-terminal region. Thus the N/C interaction does not likely involve AF1 directly thereby leaving it as an open interface. Recent studies of He et al. (80) indicate that AF2 is the AR C-terminal contact site for the N/C interaction and that p160 coactivators interact with AR through binding outside the AF2. This could explain why the AR ligand binding domain intrinsic activation function (AF2) is weak relative to AF1 of the N-terminal domain (13, 18). AR AF2 is also weaker than AF2 in other steroid receptors. In PR, for example, SRC-1 and TIF2 interact with both AF2 and AF1 (46).
In the human testis, SRC-3 coexpresses with AR and other nuclear receptors in peritubular myoid cells and Sertoli cells that mediate androgen regulation of spermatogenesis. In addition, this coactivator is expressed in spermatogenic cells that do not express AR. In these developing germ cells SRC-3 may modulate transactivation by other nuclear receptors that control spermatogenesis. Among the receptors known to interact with SRC-3 and other p160 coactivators, GR (81), ER (82, 83), RAR, and RXR (84, 85), TR (86), and VDR (87); each has a role in testicular development and/or spermatogenesis. SRC-1 is also a coactivator for other transcription factors such as AP1 (88) and serum response factor, an upstream regulatory element of c-fos (89). mRNA for c-fos increases in Sertoli cells as an immediate-early response to FSH stimulation (90).
SRC-1 knockout male mice are fertile but have a slight decrease in the testis/body weight ratio (48). An increase in TIF2 (SRC-2) mRNA in testes of SRC-1, null mice suggested this coactivator compensates partially for the loss of SRC-1. We suggest that mouse homologues of TRAM-1 and other members of the SRC-3 subgroup of p160 coactivators that include p/CIP (62) also provided compensation in the SRC-1 null mice by supporting transcriptional activation of AR and other nuclear receptors that regulate spermatogenesis.
We discovered recently that protein inhibitor of activated STAT-1
(PIAS1) is a coactivator for AR and GR (57). PIAS1 is
highly expressed in testis in a cellular distribution similar to that
of SRC-3 and Jenab and Morris (91) found that STAT-1 is
activated in Sertoli cells by the cytokines, leukemia inhibitory
factor, and interleukin-6. Thus, PIAS1 has the potential to control
multiple regulatory mechanisms in testis by mediating cross talk
between STAT-1 and AR signaling. Because activated STAT-1 utilizes CBP
and the mouse p160 acetyltransferase coactivator p/CIP (mSRC-3)
(77), which is similar to human TRAM-1, PIAS1 could
function as an integrator of STAT-1 and nuclear receptor signaling.
Another member of the PIAS family, PIASx, referred to as androgen
receptor interacting protein-3, was highly expressed in rat and human
testis and increased AR transactivation in cotransfection assays
(92). PIASx has not been reported to interact with an
activated STAT.
Other AR coregulators in testis include SNURF, a ring finger protein that bound the AR DNA-binding domain and adjoining N-terminal half of the hinge region (93). ANPK, a 130-kDa serine/threonine kinase, colocalized with AR in testis (94). AR itself was not a substrate for ANPK suggesting the coactivator function of ANPK may have resulted from the phosphorylation of AR associated regulatory proteins or components of the general transcription machinery.
In humans and other constant breeders that maintain high intratesticular levels of testosterone, AR in testicular cells is likely in a constant state of ligand activation. Thus AR transactivation in the different stages of spermatogenesis is probably determined by factors that control the levels of AR protein (5, 53) and by AR transcriptional coregulators. The multiplicity of known and likely to be discovered AR coregulators in testis reflects not only the complexity of transcription but also of spermatogenesis.
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Acknowledgments |
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Footnotes |
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Received September 8, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Nature 396:377–380[CrossRef][Medline]
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cellular retinol-binding protein, and cellular retinoic acid-binding
protein in rat testis: evidence for germ cell-specific mRNAs. Biol
Reprod 44:53–61[Abstract]
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 H.-J. Zhou, J. Yan, W. Luo, G. Ayala, S.-H. Lin, H. Erdem, M. Ittmann, S. Y. Tsai, and M.-J. Tsai SRC-3 Is Required for Prostate Cancer Cell Proliferation and Survival Cancer Res., September 1, 2005; 65(17): 7976 - 7983. [Abstract] [Full Text] [PDF]
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 K.-H. Chuang, Y.-F. Lee, W.-J. Lin, C.-Y. Chu, S. Altuwaijri, Y.-J. Y. Wan, and C. Chang 9-cis-Retinoic Acid Inhibits Androgen Receptor Activity through Activation of Retinoid X Receptor Mol. Endocrinol., May 1, 2005; 19(5): 1200 - 1212. [Abstract] [Full Text] [PDF]
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S. Bai, B. He, and E. M. Wilson Melanoma Antigen Gene Protein MAGE-11 Regulates Androgen Receptor Function by Modulating the Interdomain Interaction Mol. Cell. Biol., February 15, 2005; 25(4): 1238 - 1257. [Abstract] [Full Text] [PDF]
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 C. W. Gregory, X. Fei, L. A. Ponguta, B. He, H. M. Bill, F. S. French, and E. M. Wilson Epidermal Growth Factor Increases Coactivation of the Androgen Receptor in Recurrent Prostate Cancer J. Biol. Chem., February 20, 2004; 279(8): 7119 - 7130. [Abstract] [Full Text] [PDF]
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G. Zhou, Y. Hashimoto, I. Kwak, S. Y. Tsai, and M.-J. Tsai Role of the Steroid Receptor Coactivator SRC-3 in Cell Growth Mol. Cell. Biol., November 1, 2003; 23(21): 7742 - 7755. [Abstract] [Full Text] [PDF]
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S.-Z. Yang and S. A. Abdulkadir Early Growth Response Gene 1 Modulates Androgen Receptor Signaling in Prostate Carcinoma Cells J. Biol. Chem., October 10, 2003; 278(41): 39906 - 39911. [Abstract] [Full Text] [PDF]
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K. Nishimura, H.-J. Ting, Y. Harada, T. Tokizane, N. Nonomura, H.-Y. Kang, H.-C. Chang, S. Yeh, H. Miyamoto, M. Shin, et al. Modulation of Androgen Receptor Transactivation by Gelsolin: A Newly Identified Androgen Receptor Coregulator Cancer Res., August 15, 2003; 63(16): 4888 - 4894. [Abstract] [Full Text] [PDF]
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 Y. S. Lee, H.-J. Kim, H. J. Lee, J. W. Lee, S.-Y. Chun, S.-K. Ko, and K. Lee Activating Signal Cointegrator 1 Is Highly Expressed in Murine Testicular Leydig Cells and Enhances the Ligand-Dependent Transactivation of Androgen Receptor Biol Reprod, November 1, 2002; 67(5): 1580 - 1587. [Abstract] [Full Text] [PDF]
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J. Reid, I. Murray, K. Watt, R. Betney, and I. J. McEwan The Androgen Receptor Interacts with Multiple Regions of the Large Subunit of General Transcription Factor TFIIF J. Biol. Chem., October 18, 2002; 277(43): 41247 - 41253. [Abstract] [Full Text] [PDF]
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T. H. Thin, E. Kim, S. Yeh, E. R. Sampson, Y.-T. Chen, L. L. Collins, R. Basavappa, and C. Chang Mutations in the Helix 3 Region of the Androgen Receptor Abrogate ARA70 Promotion of 17beta -Estradiol-induced Androgen Receptor Transactivation J. Biol. Chem., September 20, 2002; 277(39): 36499 - 36508. [Abstract] [Full Text] [PDF]
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J. E. Pawlowski, J. R. Ertel, M. P. Allen, M. Xu, C. Butler, E. M. Wilson, and M. E. Wierman Liganded Androgen Receptor Interaction with beta -Catenin. NUCLEAR CO-LOCALIZATION AND MODULATION OF TRANSCRIPTIONAL ACTIVITY IN NEURONAL CELLS J. Biol. Chem., May 31, 2002; 277(23): 20702 - 20710. [Abstract] [Full Text] [PDF]
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J. Reid, S. M. Kelly, K. Watt, N. C. Price, and I. J. McEwan Conformational Analysis of the Androgen Receptor Amino-terminal Domain Involved in Transactivation. INFLUENCE OF STRUCTURE-STABILIZING SOLUTES AND PROTEIN-PROTEIN INTERACTIONS J. Biol. Chem., May 24, 2002; 277(22): 20079 - 20086. [Abstract] [Full Text] [PDF]
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J.-A. Tan, S. H. Hall, K. G. Hamil, G. Grossman, P. Petrusz, and F. S. French Protein Inhibitors of Activated STAT Resemble Scaffold Attachment Factors and Function as Interacting Nuclear Receptor Coregulators J. Biol. Chem., May 3, 2002; 277(19): 16993 - 17001. [Abstract] [Full Text] [PDF]
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C. A. Heinlein and C. Chang Androgen Receptor (AR) Coregulators: An Overview Endocr. Rev., April 1, 2002; 23(2): 175 - 200. [Abstract] [Full Text] [PDF]
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B. He, J. T. Minges, L. W. Lee, and E. M. Wilson The FXXLF Motif Mediates Androgen Receptor-specific Interactions with Coregulators J. Biol. Chem., March 15, 2002; 277(12): 10226 - 10235. [Abstract] [Full Text] [PDF]
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T. Ueda, N. Bruchovsky, and M. D. Sadar Activation of the Androgen Receptor N-terminal Domain by Interleukin-6 via MAPK and STAT3 Signal Transduction Pathways J. Biol. Chem., February 22, 2002; 277(9): 7076 - 7085. [Abstract] [Full Text] [PDF]
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Z.-x. Zhou, B. He, S. H. Hall, E. M. Wilson, and F. S. French Domain Interactions between Coregulator ARA70 and the Androgen Receptor (AR) Mol. Endocrinol., February 1, 2002; 16(2): 287 - 300. [Abstract] [Full Text] [PDF]
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J. L. Shenk, C. J. Fisher, S.-Y. Chen, X.-F. Zhou, K. Tillman, and L. Shemshedini p53 Represses Androgen-induced Transactivation of Prostate-specific Antigen by Disrupting hAR Amino- to Carboxyl-terminal Interaction J. Biol. Chem., October 12, 2001; 276(42): 38472 - 38479. [Abstract] [Full Text] [PDF]
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A. Bubulya, S.-Y. Chen, C. J. Fisher, Z. Zheng, X.-Q. Shen, and L. Shemshedini c-Jun Potentiates the Functional Interaction between the Amino and Carboxyl Termini of the Androgen Receptor J. Biol. Chem., November 21, 2001; 276(48): 44704 - 44711. [Abstract] [Full Text] [PDF]
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